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Optical Projection Tomography for Spatio-Temporal Analysis in the Zebrafish
CHAPTER 3
Optical Projection Tomography for Spatio-Temporal Analysis in the Zebrafish Robert J. Bryson-Richardson and Peter D. Currie Victor Chang Cardiac Research Institute Sydney 2010, Australia
I. II. III. IV.
V. VI.
VII. VIII. IX.
X. XI.
XII.
Introduction The Principle of Tomography Problems for OPT in Zebrafish Materials A. Reagents B. Equipment Methods A. Overview Sample Preparation A. Fixation B. Removing Pigmentation C. Mounting the Sample for Scanning Scanning Reconstruction Presentation of Reconstructions A. Surface Rendering B. Volume Rendering C. Viewing Renderings Discussion The Future of OPT A. Atlases of Morphology and Gene Expression B. Screening Tool during Larval Stages C. Time-Lapse Analysis of Embryonic Development OPT Equipment References
METHODS IN CELL BIOLOGY, VOL. 76 Copyright 2004, Elsevier Inc. All rights reserved. 0091-679X/04 $35.00
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Robert J. Bryson-Richardson and Peter D. Currie
Information about the spatio-temporal patterning of an embryonic structure, gene, or protein expression pattern can be invaluable in elucidating function. The determination of the three-dimensional (3D) organization of an anatomical structure within a zebrafish embryo has required serial confocal microscopy, followed by reconstruction, or generating a reconstruction from serial sections. The process of sectioning can damage the morphology of an embryo and cause the loss of 3D organization. Optical sectioning using confocal microscopy removes a lot of these problems in the zebrafish embryo. However, optical section in the zebrafish juvenile and adult stages is prevented by the size of the sample. In addition, for confocal analysis, samples must be fluorescently labeled precluding the analysis of many common labeling techniques such as in situ hybridization and -Galactosidase staining. To overcome these problems, we have applied the Optical projection tomography (OPT) technique described by Sharpe et al. (2002) and used it to examine phenotype and morphology in juvenile zebrafish.
I. Introduction Traditionally the analysis of gene expression and anatomy has relied on two-dimensional images, which we try to consider in the 3D context of the organism. While methods do exist for the generation of 3D images, they all have their limitations. These methods include the reconstruction of serial physical sections (Brune et al., 1999; Streicher et al., 2000; Weninger and Mohun, 2002), serial optical sections using confocal microscopy (Cooper et al., 1999), and magnetic resonance imaging (MRI). In the zebrafish, 3D analysis has largely depended on confocal imaging (Bassett et al., 2003; Isogai et al., 2001) or, to a much lesser extent, serial sectioning. Confocal microscopy requires fluorescent labeling of the sample and there are also limits on the size of the sample. Use of two-photon lasers can increase the working depth but it is not feasible to scan entire juvenile fish in this way. Generating 3D information by serial sectioning is a highly time-intensive procedure, and the resulting images do not always join to form a smooth 3D object. While MRI does not require fluorescent labeling, it also has the disadvantage that regular labeling techniques cannot be used to label tissues or expression. Additionally, MRI relies on prohibitively expensive equipment and currently has a limited resolution. A more thorough comparison of OPT to other 3D imaging techniques has been carried out by Sharpe (2003). Therefore we decided to test the method of OPT described by Sharpe et al. (2002) for the characterization of late stage morphology and also for phenotypic analysis of samples beyond the range of confocal analysis. We also tested the ability of OPT to reconstruct early stage embryos (24 h) to determine whether this method is suitable as a single tool for the analysis of zebrafish throughout development.
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II. The Principle of Tomography Tomography is the process by which information about an object is determined from the object’s sections or projections. Projection tomography has been used successfully for x-ray scans, positron emission tomography, and electron tomography. In all of these methods, a 3D object is produced from reconstructions using 2D projection images. Examining the projection of an object removes a dimension as demonstrated in Fig. 1, where the projection of a 3D sample is a 2D image. As the light travels through the sample it is reduced in intensity. Where the sample is thickest, very little light passes all the way through. Conversely, where the sample is thinnest, hardly any light is lost. The projection is like a shadow, a 3D object creating a 2D shadow. Except, in this case, the shadow is not uniformly dark but contains information about the thickness and density of the sample. From a single projection image, we cannot reconstruct the 3D shape of the object. However, if we obtain projection images from a series of angles, it is
Fig. 1 Projection of an object removes a dimension. (A) A 3D object casts a 2D shadow. In a projection, the shadow is not uniform but contains information about the object. Where the object is thickest, the shadow is darker. Similarly, where the object is thin, there is only a faint shadow. (B) Projection image from a scan of a 2-week-old zebrafish embryo. Using fluorescent imaging, a stronger signal is received from the thicker tissues and those that fluoresce at higher levels. In addition to giving information about the shape of the sample, unlike the shadow, the projection contains information about the internal structures.
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Fig. 2 Back projection. (A) Light is shone through a sample (blue) creating a projection (grey background ). The amount of light passing through the sample is dependent on the thickness of the sample. (B) The sample is rotated 90 counter-clockwise and the process is repeated. (C) To reconstruct the sample, the projection images are rotated to match the angle of the sample and merged together. This image is the result of using nine images of the sample in 22.5 intervals throughout 180 . The contrast of the image has then been adjusted to make the reconstructed object clearer. The shape of the object is clearly visible in the back projection. However, there is a star-shape pattern surrounding the object.
possible to calculate the shape of the object. The object is reconstructed as a series of 2D sections. The sections are subsequently stacked to form a 3D object. The reconstruction process used is a back projection method. In the simplest form of back projection, the projection images are projected back through the object and the combination of the images reconstructs the shape of the original image, as shown in Fig. 2. Such simple reconstruction methods have problems such as the formation of a star-shaped pattern around each object as demonstrated in Fig. 2. To remove these artifacts, a filtered back projection method is used. The Radon transform is a projective transformation that generates a projection from a 2D object. Using the inverse of the Radon transform, we can generate 2D information from the projection. The result of the inverse Radon transformation is still blurred in comparison with the original object and so filtering is carried out to reduce this eVect. A ramp filter is applied to the data either before or after back projection. The ramp filter attenuates data according to its frequency, with the greatest eVect on low-frequency data and no attenuation of the highest frequency data. The frequency refers to the rate of change of intensities in the projection (i.e., a very bright pixel next to a black pixel has a very high frequency, while a line of pixels with very similar values has a low frequency). This enhances edges in the
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reconstruction, making the results sharper. In most cases, including OPT, the filter is applied to the projection before reconstruction, as the application of a 1D filter is less computationally intensive than a 2D filter. To reconstruct the object from the series of projections, information must be obtained over at least 180 . As the number of angles used increases, the quality of the reconstruction improves, as the distance between data points is reduced.
III. Problems for OPT in Zebrafish The technique of OPT was developed using mouse embryos as samples (Sharpe et al., 2002). Obvious diVerences create problems in applying this technique to zebrafish. Early zebrafish embryos are a fraction of the size of mouse embryos. The higher magnifications used for zebrafish cause a loss of depth of field in the captured images. As a consequence of this, the sample must be orientated along the axis of rotation and carefully adjusted so that as much of the sample as possible stays in focus throughout the rotation of the sample during the scan. The best results for OPT of whole embryos have been obtained using fluorescent imaging, relying on the autofluorescence of embryonic tissue. In the zebrafish the yolk fluoresces at levels above the embryonic tissue and can reduce the quality of the reconstruction of the embryonic tissue. In antibody-labeled embryos this is not a problem as this fluorescence appears to be quenched during the staining procedure in unstained embryos. However when the morphology is being examined, a fluorescent dye must be used to increase the fluorescence of the embryonic tissue above that of the yolk. The increased fluorescence also results in short exposure times, which in turn reduce background noise in the images. When examining the early embryos and particularly the juvenile stages, pigmentation blocks the passage of light through the embryo, preventing accurate reconstruction. For this reason, where possible, we use fish with reduced pigmentation such as golden. Where this is impossible, we remove pigmentation using the method described in VI. Sample Preparation, B. Removing Pigmentation. Finally, given the smaller size of zebrafish embryonic structures, we wished to reconstruct images at the highest resolution possible. We therefore captured images approximately four times the size of those routinely used in mouse embryos. While this does result in higher resolution reconstructions, the time taken for reconstruction and subsequent rendering is greatly increased. We have therefore tried to optimize this process where possible. We describe here the technique we have developed and use for carrying out OPT on zebrafish. This application of OPT to zebrafish samples continues to be developed in our lab, and the OPT technique itself is continually being improved and refined in Edinburgh (J. Sharpe, personal communication, 2004).
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IV. Materials A. Reagents Methanol series; 100% methanol, 75% methanol, 50% methanol, 25% methanol in H2O. 2% low melting point agarose in H2O. 4% paraformaldehyde (PFA) in phosphate-buVered saline (PBS). Propidium Iodide 0.2 mg/ml. Bleach solution: 5% Formamide, 0.5 SSC, 10% H2O2. This solution is made from a 50% Formamide 5 SSC stock diluted 1 in 7 and then 3 parts 30% H2O2 added. Adding H2O2 to the undiluted formamide: SSC solution can be explosive. Benzyl alcohol : Benzyl Benzoate 2 : 1. B. Equipment 1-ml syringes with attached 30G 1/2 needles 20-ml glass vials 60- 20.3-mm Petri dishes Charge-coupled device (CCD) camera Fluorescent stereomicroscope (Leica MZ FLIII, Leica Microsystems, Gladesville, Australia) IPlab software (Scanalytics, Inc., Fairfax, VA) Macintosh Computer (OS9) Mouse Atlas software OPT reconstruction software Polyethylene gloves Reconstruction scripts MRC OPT Scanner Visualization Toolkit Unix or Linux workstation
V. Methods A. Overview To carry out OPT, the stained or unstained sample must be fixed and then mounted in agarose before dehydration. The dehydrations and subsequent clearing steps allow the light to pass through the sample. A series of 400 images of the sample are then taken throughout a 360 revolution. These 400 images can then be used to create a 3D model of the sample. This 3D model allows virtual sectioning of the sample in any plane. Alternatively, the sample can be rendered to produce images showing the surface of the sample or to give a 3D representation.
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VI. Sample Preparation A. Fixation 1. Samples should be fixed in 4% PFA at 4 C at least overnight. In young unstained zebrafish embryos, the autofluorescence of the yolk is much greater than that of embryonic tissue in unstained embryos. 2. To increase embryonic fluorescence above that of the yolk tissue, embryos younger than 72 h should be washed for 1 h at room temperature in propidium iodide at a concentration of 0.2 g/ml in PBS. 3. The embryos should be washed 5 times in H2O at room temperature.
B. Removing Pigmentation For OPT to work, light needs to be able to pass all the way through the embryo. Pigmentation therefore can reduce the quality of, or entirely prevent, reconstruction. For this reason we work, where possible, with strains with reduced or absent pigmentation such as golden. This is of course not always possible and, even in these strains, pigmentation still occurs at later stages. Therefore we remove pigmentation using a protocol similar to that described for Xenopus (Robinson and Guille, 1999). 1. After fixation, embryos are rapidly dehydrated using 100% methanol for at least 30 m at room temperature. 2. The embryos are then rehydrated through a methanol : water series of 75%, 50%, and 25% methanol and finally H2O for 10 m at each step. 3. The embryos are then transferred to the bleach solution, illuminated on a light box or microscope stage, and turned every couple of minutes. It should take 10 to 15 m for the pigmentation to disappear, the last pigmentation to go being that of the eye. In older fish the pigmentation is more diYcult to remove and may require changing the bleaching solution every 20 m until the pigmentation clears. C. Mounting the Sample for Scanning 1. Wash the embryos 4 times in H2O for 5 m to remove any traces of salts that may crystallize during the agarose mounting procedure. 2. Rinse the samples in low melting point (LMP) agarose cooled to 37 C before mounting. Mount the samples in LMP agarose in a deep 50-mm Petri dish. 3. Orient the sample within the dish using 30G 1/2 needles on a 1-ml syringe. The zebrafish should be vertical within the agarose and such that the center of the sample is approximately 1 cm from the base of the dish. To cool the LMP agarose faster, the mounting can be carried out on a bed of ice to reduce the time until the agarose sets.
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4. When the agarose starts to solidify and the sample position is fixed, move the dish to 4 C for at least 1 h to completely set. 5. Once the agarose is completely set, remove the sample using a plastic borer (made from a cut pipet). Push the borer through the agarose trying to ensure the sample is in the centre of the core to be removed. Cut away the surrounding agarose with a razor blade until the core can be removed. 6. Using a razor blade, trim the agarose to form a cone around the sample. 7. Lower the mounted sample using tweezers into a 20-ml vial containing either 25% methanol 75% H2O, for samples older than 72 h, or 100% methanol, for samples younger than 72 h and leave at 4 C overnight. The wash series is used to reduce the amount of shrinking that occurs to the sample during dehydration. If the sample shrinks more as a result of dehydration than the agarose, bubbles are seen around the sample. Samples surrounded by air bubbles cannot be accurately reconstructed. This can be reduced by longer fixation and gradual dehydration. Move samples younger than 72 h to a vial containing benzyl alcohol : benzyl benzoate (BABB) 2 : 1 over night or 5 h or more at 4 C. Move older samples through a methanol water series of 50%, 75%, 2 100% methanol for 5 h or overnight at 4 C at each step before moving to BABB overnight. Remember to wear polyethylene gloves during all procedures involving benzyl benzoate : benzyl alcohol solutions.
VII. Scanning 1. Using forceps, remove the sample from the glass vial containing BABB and transfer to a glass Petri dish. Plastic dishes can be used but degrade with exposure to BABB. The excess BABB should be removed using tissue and then the sample transferred to the OPT machine. It is best to move the sample in the dish to the machine and then transfer to the OPT scanner to limit the chance of dropping BABB on to the scanner or microscope. If any BABB is spilled, it should be removed immediately to prevent corrosion of the scanner. 2. Once the sample is in the scanner and ready to be scanned, run the OPT scanner software. This guides the user through the process of adjusting the position of the mount, ensuring the specimen remains within the field of view, and is as close as possible to the axis of rotation. Then set the exposure and focus to capture the 400 images throughout the 360 rotation. The OPT scanner software is used in combination with IPlab (Scanalytics, Fairfax, VA). Images can be captured at either the full resolution of the camera (for example 1360 1036 pixels) or binning 2, depending on the resolution required. While the results obtained without binning are of a higher
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resolution, there is an increase in the scanning times and a large increase in the time required for all the subsequent reconstruction steps. 3. Save the complete image series as a 12-bit tiV stack and then copied to a CD or DVD for backup and transfer to the Unix workstation used for reconstruction. 4. Where possible, crop the image stack to remove unnecessary blank space around the sample as this reduces the size of the images and significantly reduces the reconstruction times. It is common that the sample will only fill the center of the image, given the shape of fish. When cropping the images, a small space should be left around the sample to prevent edge eVects forming too close to the sample.
VIII. Reconstruction Reconstruction of the data is carried out on a Unix or Linux workstation. The required software is written by the Edinburgh Mouse Atlas project (Baldock et al., 2003) and the Sharpe lab. These programs are run from the command line and require a basic understanding of Unix. The OPT software copies the data to the Unix system and converts the images to the wlz format used by the reconstruction software. Wlz format images can be viewed using the MA3Dview software. 1. The images are analyzed and noise and bad pixels removed using OptPreprocess. The images are then ready to be reconstructed by the OptRecon program. 2. The OptRecon program assumes that the axis of rotation is in the center of the image, which is not always the case. To determine the displacement of the axis of rotation from the center, a series of individual sections throughout the sample are reconstructed using OptRecon to test diVerent displacement values. If an incorrect value is used, the image will become blurred, and objects start to appear as circles as opposed to discrete spots. The optimum value is determined by empirical examination of the reconstructed sections created using a range of displacement values. Once the optimum value has been determined, the entire object is reconstructed using this value. 3. The reconstructed object is a 16-bit grey object. The grey range can be adjusted to reduce background noise and be converted to an 8-bit object using MA3Dview. The object is also cropped to remove excess blank space from around the sample and to reduce the file sizes further still. The resulting object is the end result of the reconstruction process, sections of the object in any plane be can be viewed using the MA3Dview software (Fig. 3). The process of reconstruction is summarized in Fig. 4.
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Fig. 3 Parasagittal section through a reconstruction of a 2-week-old juvenile. Many of the organs and tissues are clearly defined, including the heart where the atrium (A), ventricle (V), and bulbus arteriosus (BA) are visible. Further demonstrating the high resolution obtained, individual muscle fibers are discernable within the myotome.
IX. Presentation of Reconstructions Using MA3Dview software developed by the Edinburgh Mouse Atlas project, the completed reconstruction can be viewed as a series of 2D sections. The data can also now be visualized in 3D by applying surface or volume rendering techniques. The output from the OPT is a digital 3D object. One of the advantages of this type of data is that it can be visualized in many ways, some of which we present later in this chapter. We can also use rendering techniques to create isosurface models or volume models. Although a thorough description of rendering techniques and software is beyond the scope of this chapter, we present examples of these methods. A. Surface Rendering The first step is to convert the 3D object to the .slc format, using the WlzExtFFConvert program. The .slc format stores the 3D object as a series of contours. Surface rendering produces a solid-appearing object by linking voxels with a gray value above a certain threshold. We use the IsoSurface program that is part of the Visualization Tool Kit (VTK; Schroeder et al., 2003). This creates a surfacerendered object that can be visualized and manipulated using vtkDecimate. For example, if all values in a 3D object of a zebrafish were above 20, creating an
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Fig. 4 The process of reconstruction. The major steps in the reconstruction process are shown together with the programs used at those stages (italicized in parenthesis).
isosurface with a threshold of 20 would produce a model of the surface of the whole fish in 3D. If the sample had been labeled with a neuronal-specific antibody, the values in the neurons would be much higher (e.g., 200). The creation of a surface rendering with a cutoV of 200 would therefore produce a model of just the neurons. By reducing the opacity of the model of the whole fish, we can merge the two renderings and have a model showing the 3D pattern within the context of the sample (Fig. 5A).
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Fig. 5 Volume and surface renderings. (A) Surface rendering of 28-somite stage embryo labeled with an antiacetylated antibody. The 3D reconstruction can be surface rendered to give a better visualization of the 3D object. The rendered object can be rotated to allow any angle to be viewed. Thresholding can be used to identify the antibody signal (green) over that of the embryo. Acetylated tubulin is one of the first components of the neuronal microtubule cytoskeleton to form and is a marker of developing post-mitotic neurons (Piperno and Fuller, 1985; Wilson and Easter, Jr., 1991). (B) Volume rendering of a reconstruction of a 2-week-old juvenile zebrafish. Like surface rendering, volume rendering can be used to give a clear 3D representation of the sample. By reducing the opacity of the rendered object, all of the internal features can be visualized.
B. Volume Rendering The VTK also provides tools for generating volume renderings. Again, using WlzExtFFConvert, the first step is to convert the 3D object to the .slc format. This model can then be viewed using scripts that use functions from VTK, such as volRen. As for surface renderings, thresholds can be applied to remove background noise. The data set is rendered with a reduced opacity so that all of the internal features are still visible compared with surface renderings where a hollow shell representing the shape of embryo is produced (Fig. 5B). C. Viewing Renderings While 2D images of these renderings are useful to display the 3D patterns, the best display of a 3D object is achieved when the object can be moved in a movie or by using the software described earlier in this chapter. In addition to direct visualization of these rendered objects, a series of 2D rendered images may be
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combined to form a movie, which is a convenient way to visualize and distribute the data without specialized software or knowledge.
X. Discussion We have demonstrated that OPT can be used on zebrafish as small as 26-somite embryos and up to 2-week-old juvenile fish, providing results that are of suYcient quality to present valuable information about a staining pattern or anatomical features. The visualization of morphology of juvenile fish has previously only been possible using serial sectioning, OPT provides a nondestructive method for obtaining the morphology of the sample and additionally provides a complete 3D object that can be further manipulated and rendered.
XI. The Future of OPT A. Atlases of Morphology and Gene Expression OPT allows atlases of morphology and gene-expression pattern to be easily generated in a similar way to data that is currently being collected for the mouse. In addition to morphological and phenotypic characterization of zebrafish, these techniques for the generation of high-resolution 3D models at various stages of development will provide valuable staging series and atlases for other species. As only a single embryo would be required at each stage, this would be even more beneficial in the study of organisms where embryonic samples are diYcult to obtain. B. Screening Tool during Larval Stages The large quantities of information that can be extracted from a single embryo means that OPT is suitable for use in high-throughput screens, characterizing phenotypes or expression patterns at embryonic and larval stages. Recording 3D data from a single embryo prevents the need for repeated sectioning, improving the speed at which screens can be carried out and greatly aiding the analysis of the results. In addition to its use for description, OPT also produces quantitative data in the intensity levels of expression. It may be possible to use this data to compare levels of expression between diVerent tissues in a quantitative manner. C. Time-Lapse Analysis of Embryonic Development Given the optical clarity of the early embryo and the ability for embryos to be mounted alive in agarose, it may be possible to image live embryos in 3D throughout development. Although diVerences in the optical properties of embryonic tissues have currently prevented such an approach, tomographic principles do exist that may allow the imaging of diVracting samples.
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XII. OPT Equipment The OPT technology was invented by J. Sharpe at the MRC Human Genetics Unit in Edinburgh, Scotland, and we performed this work through a collaboration. The MRC is currently redesigning and commercializing the technique (as a fully integrated imaging system), so please contact the Sharpe lab for any requests ([email protected]). References Baldock, R. A., Bard, J. B., Burger, A., Burton, N., Christiansen, J., Feng, G., Hill, B., Houghton, D., Kaufman, M., Rao, J., et al. (2003). EMAP and EMAGE: A framework for understanding spatially organized data. Neuroinformatics 1, 309–325. Bassett, D. I., Bryson-Richardson, R. J., Daggett, D. F., Gautier, P., Keenan, D. G., and Currie, P. D. (2003). Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo. Development 130, 5851–5860. Brune, R. M., Bard, J. B., Dubreuil, C., Guest, E., Hill, W., Kaufman, M., Stark, M., Davidson, D., and Baldock, R. A. (1999). A three-dimensional model of the mouse at embryonic day 9. Dev. Biol. 216, 457–468. Cooper, M. S., D’Amico, L. A., and Henry, C. A. (1999). Confocal microscopic analysis of morphogenetic movements. In ‘‘Methods in Cell Biology’’ (H. W. Detrich, M. Westerfield, and L. I. Zon, eds.), pp. 179–204. Academic Press, San Diego. Isogai, S., Horiguchi, M., and Weinstein, B. M. (2001). The vascular anatomy of the developing zebrafish: An atlas of embryonic and early larval development. Dev. Biol. 230, 278–301. Piperno, G., and Fuller, M. T. (1985). Monoclonal antibodies specific for an acetylated form of alphatubulin recognize the antigen in cilia and flagella from a variety of organisms. J. Cell Biol. 101, 2085–2094. Robinson, C., and Guille, M. (1999). Immunohistochemistry of Xenopus embryos. In ‘‘Methods in Molecular Biology’’ (M. Guille, ed.), pp. 89–97. Humana Press, Totowa, NJ. Schroeder, W., Martin, K., and Lorenson, B. (2003). ‘‘The Visualization Toolkit: An Object Oriented Approach to 3D Graphics.’’ Kitware Inc., New York. Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A., Hecksher-Sorensen, J., Baldock, R., and Davidson, D. (2002). Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science 296, 541–545. Sharpe, J. (2003). Optical projection tomography as a new tool for studying embryo anatomy. J. Anat. 202, 175–181. Streicher, J., Donat, M. A., Strauss, B., Sporle, R., Schughart, K., and Muller, G. B. (2000). Computer-based three-dimensional visualization of developmental gene expression. Nat. Genet. 25, 147–152. Weninger, W. J., and Mohun, T. (2002). Phenotyping transgenic embryos: A rapid 3-D screening method based on episcopic fluorescence image capturing. Nat. Genet. 30, 59–65. Wilson, S. W., and Easter, S. S., Jr. (1991). Stereotyped pathway selection by growth cones of early epiphysial neurons in the embryonic zebrafish. Development 112, 723–746.
Optical Projection Tomography for Spatio-Temporal Analysis in the Zebrafish Robert J. Bryson-Richardson and Peter D. Currie Victor Chang Cardiac Research Institute Sydney 2010, Australia
I. II. III. IV.
V. VI.
VII. VIII. IX.
X. XI.
XII.
Introduction The Principle of Tomography Problems for OPT in Zebrafish Materials A. Reagents B. Equipment Methods A. Overview Sample Preparation A. Fixation B. Removing Pigmentation C. Mounting the Sample for Scanning Scanning Reconstruction Presentation of Reconstructions A. Surface Rendering B. Volume Rendering C. Viewing Renderings Discussion The Future of OPT A. Atlases of Morphology and Gene Expression B. Screening Tool during Larval Stages C. Time-Lapse Analysis of Embryonic Development OPT Equipment References
METHODS IN CELL BIOLOGY, VOL. 76 Copyright 2004, Elsevier Inc. All rights reserved. 0091-679X/04 $35.00
37
38
Robert J. Bryson-Richardson and Peter D. Currie
Information about the spatio-temporal patterning of an embryonic structure, gene, or protein expression pattern can be invaluable in elucidating function. The determination of the three-dimensional (3D) organization of an anatomical structure within a zebrafish embryo has required serial confocal microscopy, followed by reconstruction, or generating a reconstruction from serial sections. The process of sectioning can damage the morphology of an embryo and cause the loss of 3D organization. Optical sectioning using confocal microscopy removes a lot of these problems in the zebrafish embryo. However, optical section in the zebrafish juvenile and adult stages is prevented by the size of the sample. In addition, for confocal analysis, samples must be fluorescently labeled precluding the analysis of many common labeling techniques such as in situ hybridization and -Galactosidase staining. To overcome these problems, we have applied the Optical projection tomography (OPT) technique described by Sharpe et al. (2002) and used it to examine phenotype and morphology in juvenile zebrafish.
I. Introduction Traditionally the analysis of gene expression and anatomy has relied on two-dimensional images, which we try to consider in the 3D context of the organism. While methods do exist for the generation of 3D images, they all have their limitations. These methods include the reconstruction of serial physical sections (Brune et al., 1999; Streicher et al., 2000; Weninger and Mohun, 2002), serial optical sections using confocal microscopy (Cooper et al., 1999), and magnetic resonance imaging (MRI). In the zebrafish, 3D analysis has largely depended on confocal imaging (Bassett et al., 2003; Isogai et al., 2001) or, to a much lesser extent, serial sectioning. Confocal microscopy requires fluorescent labeling of the sample and there are also limits on the size of the sample. Use of two-photon lasers can increase the working depth but it is not feasible to scan entire juvenile fish in this way. Generating 3D information by serial sectioning is a highly time-intensive procedure, and the resulting images do not always join to form a smooth 3D object. While MRI does not require fluorescent labeling, it also has the disadvantage that regular labeling techniques cannot be used to label tissues or expression. Additionally, MRI relies on prohibitively expensive equipment and currently has a limited resolution. A more thorough comparison of OPT to other 3D imaging techniques has been carried out by Sharpe (2003). Therefore we decided to test the method of OPT described by Sharpe et al. (2002) for the characterization of late stage morphology and also for phenotypic analysis of samples beyond the range of confocal analysis. We also tested the ability of OPT to reconstruct early stage embryos (24 h) to determine whether this method is suitable as a single tool for the analysis of zebrafish throughout development.
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II. The Principle of Tomography Tomography is the process by which information about an object is determined from the object’s sections or projections. Projection tomography has been used successfully for x-ray scans, positron emission tomography, and electron tomography. In all of these methods, a 3D object is produced from reconstructions using 2D projection images. Examining the projection of an object removes a dimension as demonstrated in Fig. 1, where the projection of a 3D sample is a 2D image. As the light travels through the sample it is reduced in intensity. Where the sample is thickest, very little light passes all the way through. Conversely, where the sample is thinnest, hardly any light is lost. The projection is like a shadow, a 3D object creating a 2D shadow. Except, in this case, the shadow is not uniformly dark but contains information about the thickness and density of the sample. From a single projection image, we cannot reconstruct the 3D shape of the object. However, if we obtain projection images from a series of angles, it is
Fig. 1 Projection of an object removes a dimension. (A) A 3D object casts a 2D shadow. In a projection, the shadow is not uniform but contains information about the object. Where the object is thickest, the shadow is darker. Similarly, where the object is thin, there is only a faint shadow. (B) Projection image from a scan of a 2-week-old zebrafish embryo. Using fluorescent imaging, a stronger signal is received from the thicker tissues and those that fluoresce at higher levels. In addition to giving information about the shape of the sample, unlike the shadow, the projection contains information about the internal structures.
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Fig. 2 Back projection. (A) Light is shone through a sample (blue) creating a projection (grey background ). The amount of light passing through the sample is dependent on the thickness of the sample. (B) The sample is rotated 90 counter-clockwise and the process is repeated. (C) To reconstruct the sample, the projection images are rotated to match the angle of the sample and merged together. This image is the result of using nine images of the sample in 22.5 intervals throughout 180 . The contrast of the image has then been adjusted to make the reconstructed object clearer. The shape of the object is clearly visible in the back projection. However, there is a star-shape pattern surrounding the object.
possible to calculate the shape of the object. The object is reconstructed as a series of 2D sections. The sections are subsequently stacked to form a 3D object. The reconstruction process used is a back projection method. In the simplest form of back projection, the projection images are projected back through the object and the combination of the images reconstructs the shape of the original image, as shown in Fig. 2. Such simple reconstruction methods have problems such as the formation of a star-shaped pattern around each object as demonstrated in Fig. 2. To remove these artifacts, a filtered back projection method is used. The Radon transform is a projective transformation that generates a projection from a 2D object. Using the inverse of the Radon transform, we can generate 2D information from the projection. The result of the inverse Radon transformation is still blurred in comparison with the original object and so filtering is carried out to reduce this eVect. A ramp filter is applied to the data either before or after back projection. The ramp filter attenuates data according to its frequency, with the greatest eVect on low-frequency data and no attenuation of the highest frequency data. The frequency refers to the rate of change of intensities in the projection (i.e., a very bright pixel next to a black pixel has a very high frequency, while a line of pixels with very similar values has a low frequency). This enhances edges in the
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reconstruction, making the results sharper. In most cases, including OPT, the filter is applied to the projection before reconstruction, as the application of a 1D filter is less computationally intensive than a 2D filter. To reconstruct the object from the series of projections, information must be obtained over at least 180 . As the number of angles used increases, the quality of the reconstruction improves, as the distance between data points is reduced.
III. Problems for OPT in Zebrafish The technique of OPT was developed using mouse embryos as samples (Sharpe et al., 2002). Obvious diVerences create problems in applying this technique to zebrafish. Early zebrafish embryos are a fraction of the size of mouse embryos. The higher magnifications used for zebrafish cause a loss of depth of field in the captured images. As a consequence of this, the sample must be orientated along the axis of rotation and carefully adjusted so that as much of the sample as possible stays in focus throughout the rotation of the sample during the scan. The best results for OPT of whole embryos have been obtained using fluorescent imaging, relying on the autofluorescence of embryonic tissue. In the zebrafish the yolk fluoresces at levels above the embryonic tissue and can reduce the quality of the reconstruction of the embryonic tissue. In antibody-labeled embryos this is not a problem as this fluorescence appears to be quenched during the staining procedure in unstained embryos. However when the morphology is being examined, a fluorescent dye must be used to increase the fluorescence of the embryonic tissue above that of the yolk. The increased fluorescence also results in short exposure times, which in turn reduce background noise in the images. When examining the early embryos and particularly the juvenile stages, pigmentation blocks the passage of light through the embryo, preventing accurate reconstruction. For this reason, where possible, we use fish with reduced pigmentation such as golden. Where this is impossible, we remove pigmentation using the method described in VI. Sample Preparation, B. Removing Pigmentation. Finally, given the smaller size of zebrafish embryonic structures, we wished to reconstruct images at the highest resolution possible. We therefore captured images approximately four times the size of those routinely used in mouse embryos. While this does result in higher resolution reconstructions, the time taken for reconstruction and subsequent rendering is greatly increased. We have therefore tried to optimize this process where possible. We describe here the technique we have developed and use for carrying out OPT on zebrafish. This application of OPT to zebrafish samples continues to be developed in our lab, and the OPT technique itself is continually being improved and refined in Edinburgh (J. Sharpe, personal communication, 2004).
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IV. Materials A. Reagents Methanol series; 100% methanol, 75% methanol, 50% methanol, 25% methanol in H2O. 2% low melting point agarose in H2O. 4% paraformaldehyde (PFA) in phosphate-buVered saline (PBS). Propidium Iodide 0.2 mg/ml. Bleach solution: 5% Formamide, 0.5 SSC, 10% H2O2. This solution is made from a 50% Formamide 5 SSC stock diluted 1 in 7 and then 3 parts 30% H2O2 added. Adding H2O2 to the undiluted formamide: SSC solution can be explosive. Benzyl alcohol : Benzyl Benzoate 2 : 1. B. Equipment 1-ml syringes with attached 30G 1/2 needles 20-ml glass vials 60- 20.3-mm Petri dishes Charge-coupled device (CCD) camera Fluorescent stereomicroscope (Leica MZ FLIII, Leica Microsystems, Gladesville, Australia) IPlab software (Scanalytics, Inc., Fairfax, VA) Macintosh Computer (OS9) Mouse Atlas software OPT reconstruction software Polyethylene gloves Reconstruction scripts MRC OPT Scanner Visualization Toolkit Unix or Linux workstation
V. Methods A. Overview To carry out OPT, the stained or unstained sample must be fixed and then mounted in agarose before dehydration. The dehydrations and subsequent clearing steps allow the light to pass through the sample. A series of 400 images of the sample are then taken throughout a 360 revolution. These 400 images can then be used to create a 3D model of the sample. This 3D model allows virtual sectioning of the sample in any plane. Alternatively, the sample can be rendered to produce images showing the surface of the sample or to give a 3D representation.
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VI. Sample Preparation A. Fixation 1. Samples should be fixed in 4% PFA at 4 C at least overnight. In young unstained zebrafish embryos, the autofluorescence of the yolk is much greater than that of embryonic tissue in unstained embryos. 2. To increase embryonic fluorescence above that of the yolk tissue, embryos younger than 72 h should be washed for 1 h at room temperature in propidium iodide at a concentration of 0.2 g/ml in PBS. 3. The embryos should be washed 5 times in H2O at room temperature.
B. Removing Pigmentation For OPT to work, light needs to be able to pass all the way through the embryo. Pigmentation therefore can reduce the quality of, or entirely prevent, reconstruction. For this reason we work, where possible, with strains with reduced or absent pigmentation such as golden. This is of course not always possible and, even in these strains, pigmentation still occurs at later stages. Therefore we remove pigmentation using a protocol similar to that described for Xenopus (Robinson and Guille, 1999). 1. After fixation, embryos are rapidly dehydrated using 100% methanol for at least 30 m at room temperature. 2. The embryos are then rehydrated through a methanol : water series of 75%, 50%, and 25% methanol and finally H2O for 10 m at each step. 3. The embryos are then transferred to the bleach solution, illuminated on a light box or microscope stage, and turned every couple of minutes. It should take 10 to 15 m for the pigmentation to disappear, the last pigmentation to go being that of the eye. In older fish the pigmentation is more diYcult to remove and may require changing the bleaching solution every 20 m until the pigmentation clears. C. Mounting the Sample for Scanning 1. Wash the embryos 4 times in H2O for 5 m to remove any traces of salts that may crystallize during the agarose mounting procedure. 2. Rinse the samples in low melting point (LMP) agarose cooled to 37 C before mounting. Mount the samples in LMP agarose in a deep 50-mm Petri dish. 3. Orient the sample within the dish using 30G 1/2 needles on a 1-ml syringe. The zebrafish should be vertical within the agarose and such that the center of the sample is approximately 1 cm from the base of the dish. To cool the LMP agarose faster, the mounting can be carried out on a bed of ice to reduce the time until the agarose sets.
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4. When the agarose starts to solidify and the sample position is fixed, move the dish to 4 C for at least 1 h to completely set. 5. Once the agarose is completely set, remove the sample using a plastic borer (made from a cut pipet). Push the borer through the agarose trying to ensure the sample is in the centre of the core to be removed. Cut away the surrounding agarose with a razor blade until the core can be removed. 6. Using a razor blade, trim the agarose to form a cone around the sample. 7. Lower the mounted sample using tweezers into a 20-ml vial containing either 25% methanol 75% H2O, for samples older than 72 h, or 100% methanol, for samples younger than 72 h and leave at 4 C overnight. The wash series is used to reduce the amount of shrinking that occurs to the sample during dehydration. If the sample shrinks more as a result of dehydration than the agarose, bubbles are seen around the sample. Samples surrounded by air bubbles cannot be accurately reconstructed. This can be reduced by longer fixation and gradual dehydration. Move samples younger than 72 h to a vial containing benzyl alcohol : benzyl benzoate (BABB) 2 : 1 over night or 5 h or more at 4 C. Move older samples through a methanol water series of 50%, 75%, 2 100% methanol for 5 h or overnight at 4 C at each step before moving to BABB overnight. Remember to wear polyethylene gloves during all procedures involving benzyl benzoate : benzyl alcohol solutions.
VII. Scanning 1. Using forceps, remove the sample from the glass vial containing BABB and transfer to a glass Petri dish. Plastic dishes can be used but degrade with exposure to BABB. The excess BABB should be removed using tissue and then the sample transferred to the OPT machine. It is best to move the sample in the dish to the machine and then transfer to the OPT scanner to limit the chance of dropping BABB on to the scanner or microscope. If any BABB is spilled, it should be removed immediately to prevent corrosion of the scanner. 2. Once the sample is in the scanner and ready to be scanned, run the OPT scanner software. This guides the user through the process of adjusting the position of the mount, ensuring the specimen remains within the field of view, and is as close as possible to the axis of rotation. Then set the exposure and focus to capture the 400 images throughout the 360 rotation. The OPT scanner software is used in combination with IPlab (Scanalytics, Fairfax, VA). Images can be captured at either the full resolution of the camera (for example 1360 1036 pixels) or binning 2, depending on the resolution required. While the results obtained without binning are of a higher
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resolution, there is an increase in the scanning times and a large increase in the time required for all the subsequent reconstruction steps. 3. Save the complete image series as a 12-bit tiV stack and then copied to a CD or DVD for backup and transfer to the Unix workstation used for reconstruction. 4. Where possible, crop the image stack to remove unnecessary blank space around the sample as this reduces the size of the images and significantly reduces the reconstruction times. It is common that the sample will only fill the center of the image, given the shape of fish. When cropping the images, a small space should be left around the sample to prevent edge eVects forming too close to the sample.
VIII. Reconstruction Reconstruction of the data is carried out on a Unix or Linux workstation. The required software is written by the Edinburgh Mouse Atlas project (Baldock et al., 2003) and the Sharpe lab. These programs are run from the command line and require a basic understanding of Unix. The OPT software copies the data to the Unix system and converts the images to the wlz format used by the reconstruction software. Wlz format images can be viewed using the MA3Dview software. 1. The images are analyzed and noise and bad pixels removed using OptPreprocess. The images are then ready to be reconstructed by the OptRecon program. 2. The OptRecon program assumes that the axis of rotation is in the center of the image, which is not always the case. To determine the displacement of the axis of rotation from the center, a series of individual sections throughout the sample are reconstructed using OptRecon to test diVerent displacement values. If an incorrect value is used, the image will become blurred, and objects start to appear as circles as opposed to discrete spots. The optimum value is determined by empirical examination of the reconstructed sections created using a range of displacement values. Once the optimum value has been determined, the entire object is reconstructed using this value. 3. The reconstructed object is a 16-bit grey object. The grey range can be adjusted to reduce background noise and be converted to an 8-bit object using MA3Dview. The object is also cropped to remove excess blank space from around the sample and to reduce the file sizes further still. The resulting object is the end result of the reconstruction process, sections of the object in any plane be can be viewed using the MA3Dview software (Fig. 3). The process of reconstruction is summarized in Fig. 4.
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Fig. 3 Parasagittal section through a reconstruction of a 2-week-old juvenile. Many of the organs and tissues are clearly defined, including the heart where the atrium (A), ventricle (V), and bulbus arteriosus (BA) are visible. Further demonstrating the high resolution obtained, individual muscle fibers are discernable within the myotome.
IX. Presentation of Reconstructions Using MA3Dview software developed by the Edinburgh Mouse Atlas project, the completed reconstruction can be viewed as a series of 2D sections. The data can also now be visualized in 3D by applying surface or volume rendering techniques. The output from the OPT is a digital 3D object. One of the advantages of this type of data is that it can be visualized in many ways, some of which we present later in this chapter. We can also use rendering techniques to create isosurface models or volume models. Although a thorough description of rendering techniques and software is beyond the scope of this chapter, we present examples of these methods. A. Surface Rendering The first step is to convert the 3D object to the .slc format, using the WlzExtFFConvert program. The .slc format stores the 3D object as a series of contours. Surface rendering produces a solid-appearing object by linking voxels with a gray value above a certain threshold. We use the IsoSurface program that is part of the Visualization Tool Kit (VTK; Schroeder et al., 2003). This creates a surfacerendered object that can be visualized and manipulated using vtkDecimate. For example, if all values in a 3D object of a zebrafish were above 20, creating an
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Fig. 4 The process of reconstruction. The major steps in the reconstruction process are shown together with the programs used at those stages (italicized in parenthesis).
isosurface with a threshold of 20 would produce a model of the surface of the whole fish in 3D. If the sample had been labeled with a neuronal-specific antibody, the values in the neurons would be much higher (e.g., 200). The creation of a surface rendering with a cutoV of 200 would therefore produce a model of just the neurons. By reducing the opacity of the model of the whole fish, we can merge the two renderings and have a model showing the 3D pattern within the context of the sample (Fig. 5A).
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Fig. 5 Volume and surface renderings. (A) Surface rendering of 28-somite stage embryo labeled with an antiacetylated antibody. The 3D reconstruction can be surface rendered to give a better visualization of the 3D object. The rendered object can be rotated to allow any angle to be viewed. Thresholding can be used to identify the antibody signal (green) over that of the embryo. Acetylated tubulin is one of the first components of the neuronal microtubule cytoskeleton to form and is a marker of developing post-mitotic neurons (Piperno and Fuller, 1985; Wilson and Easter, Jr., 1991). (B) Volume rendering of a reconstruction of a 2-week-old juvenile zebrafish. Like surface rendering, volume rendering can be used to give a clear 3D representation of the sample. By reducing the opacity of the rendered object, all of the internal features can be visualized.
B. Volume Rendering The VTK also provides tools for generating volume renderings. Again, using WlzExtFFConvert, the first step is to convert the 3D object to the .slc format. This model can then be viewed using scripts that use functions from VTK, such as volRen. As for surface renderings, thresholds can be applied to remove background noise. The data set is rendered with a reduced opacity so that all of the internal features are still visible compared with surface renderings where a hollow shell representing the shape of embryo is produced (Fig. 5B). C. Viewing Renderings While 2D images of these renderings are useful to display the 3D patterns, the best display of a 3D object is achieved when the object can be moved in a movie or by using the software described earlier in this chapter. In addition to direct visualization of these rendered objects, a series of 2D rendered images may be
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combined to form a movie, which is a convenient way to visualize and distribute the data without specialized software or knowledge.
X. Discussion We have demonstrated that OPT can be used on zebrafish as small as 26-somite embryos and up to 2-week-old juvenile fish, providing results that are of suYcient quality to present valuable information about a staining pattern or anatomical features. The visualization of morphology of juvenile fish has previously only been possible using serial sectioning, OPT provides a nondestructive method for obtaining the morphology of the sample and additionally provides a complete 3D object that can be further manipulated and rendered.
XI. The Future of OPT A. Atlases of Morphology and Gene Expression OPT allows atlases of morphology and gene-expression pattern to be easily generated in a similar way to data that is currently being collected for the mouse. In addition to morphological and phenotypic characterization of zebrafish, these techniques for the generation of high-resolution 3D models at various stages of development will provide valuable staging series and atlases for other species. As only a single embryo would be required at each stage, this would be even more beneficial in the study of organisms where embryonic samples are diYcult to obtain. B. Screening Tool during Larval Stages The large quantities of information that can be extracted from a single embryo means that OPT is suitable for use in high-throughput screens, characterizing phenotypes or expression patterns at embryonic and larval stages. Recording 3D data from a single embryo prevents the need for repeated sectioning, improving the speed at which screens can be carried out and greatly aiding the analysis of the results. In addition to its use for description, OPT also produces quantitative data in the intensity levels of expression. It may be possible to use this data to compare levels of expression between diVerent tissues in a quantitative manner. C. Time-Lapse Analysis of Embryonic Development Given the optical clarity of the early embryo and the ability for embryos to be mounted alive in agarose, it may be possible to image live embryos in 3D throughout development. Although diVerences in the optical properties of embryonic tissues have currently prevented such an approach, tomographic principles do exist that may allow the imaging of diVracting samples.
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XII. OPT Equipment The OPT technology was invented by J. Sharpe at the MRC Human Genetics Unit in Edinburgh, Scotland, and we performed this work through a collaboration. The MRC is currently redesigning and commercializing the technique (as a fully integrated imaging system), so please contact the Sharpe lab for any requests ([email protected]). References Baldock, R. A., Bard, J. B., Burger, A., Burton, N., Christiansen, J., Feng, G., Hill, B., Houghton, D., Kaufman, M., Rao, J., et al. (2003). EMAP and EMAGE: A framework for understanding spatially organized data. Neuroinformatics 1, 309–325. Bassett, D. I., Bryson-Richardson, R. J., Daggett, D. F., Gautier, P., Keenan, D. G., and Currie, P. D. (2003). Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo. Development 130, 5851–5860. Brune, R. M., Bard, J. B., Dubreuil, C., Guest, E., Hill, W., Kaufman, M., Stark, M., Davidson, D., and Baldock, R. A. (1999). A three-dimensional model of the mouse at embryonic day 9. Dev. Biol. 216, 457–468. Cooper, M. S., D’Amico, L. A., and Henry, C. A. (1999). Confocal microscopic analysis of morphogenetic movements. In ‘‘Methods in Cell Biology’’ (H. W. Detrich, M. Westerfield, and L. I. Zon, eds.), pp. 179–204. Academic Press, San Diego. Isogai, S., Horiguchi, M., and Weinstein, B. M. (2001). The vascular anatomy of the developing zebrafish: An atlas of embryonic and early larval development. Dev. Biol. 230, 278–301. Piperno, G., and Fuller, M. T. (1985). Monoclonal antibodies specific for an acetylated form of alphatubulin recognize the antigen in cilia and flagella from a variety of organisms. J. Cell Biol. 101, 2085–2094. Robinson, C., and Guille, M. (1999). Immunohistochemistry of Xenopus embryos. In ‘‘Methods in Molecular Biology’’ (M. Guille, ed.), pp. 89–97. Humana Press, Totowa, NJ. Schroeder, W., Martin, K., and Lorenson, B. (2003). ‘‘The Visualization Toolkit: An Object Oriented Approach to 3D Graphics.’’ Kitware Inc., New York. Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A., Hecksher-Sorensen, J., Baldock, R., and Davidson, D. (2002). Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science 296, 541–545. Sharpe, J. (2003). Optical projection tomography as a new tool for studying embryo anatomy. J. Anat. 202, 175–181. Streicher, J., Donat, M. A., Strauss, B., Sporle, R., Schughart, K., and Muller, G. B. (2000). Computer-based three-dimensional visualization of developmental gene expression. Nat. Genet. 25, 147–152. Weninger, W. J., and Mohun, T. (2002). Phenotyping transgenic embryos: A rapid 3-D screening method based on episcopic fluorescence image capturing. Nat. Genet. 30, 59–65. Wilson, S. W., and Easter, S. S., Jr. (1991). Stereotyped pathway selection by growth cones of early epiphysial neurons in the embryonic zebrafish. Development 112, 723–746.